The initiation of blood clotting is by two different, yet similar, molecular mechanisms called the intrinsic and extrinsic coagulation pathways, or cascades. The intrinsic pathway involves factors that are normally in the blood. The extrinsic pathway involves tissue factors in addition to blood components. In each of the reaction steps of the two cascades, a proteinase converts an inactive zymogen into its enzymically active form. In the last step of the cascade, which is the same in both the intrinsic and extrinsic pathways, inactive prothrombin is converted to thrombin, which, in turn, catalyzes the conversion of soluble fibrinogen into insoluble fibrin.
The conversions of zymogens into active proteinases in both cascades are extremely slow in the absence of accessory factors, which stimulate the rates of zymogen activation from 10.sup.4 to 10.sup.5 times the rates observed in their absence. Three kinds of accessory factors operate in the cascades: (1) Ca.sup.2+ ions, (2) acidic phospholipids derived from the membrane bilayers of platelets or damaged tissues, and (3) a protein cofactor that is specifically required for activation of a given zymogen. Prothrombin, which has a molecular weight of about 69,000 to about 80,000, is composed of a single polypeptide chain. Like the activation of all blood coagulation zymogens, activation of prothrombin occurs on a surface provided by negatively-charged phospholipids, such as phosphatidylserine, which are from platelets or damaged tissue. Such lipids are found almost exclusively on the cytoplasmic side, or inside, of the lipid bilayers of cell membranes. Prothrombin does not adhere to erythrocytes or endothelial cells of the vascular system unless the cells are disrupted to expose their inner surfaces. Thus, disruption of cells to expose negatively-charged phospholipids permits prothrombin to bind to the phospholipids. This is a first step in the activation of prothrombin. The binding of prothrombin to the phospholipids is a Ca.sup.2+ -dependent process.
The activation of prothrombin is catalyzed by Factor X.sub.a, which also binds to phospholipids through Ca.sup.2+ -dependent interactions. However, maximal rates of activation of prothrombin are obtained only if Factor V.sub.a is also bound to the prothrombin-X.sub.a -Ca.sup.2+ -phospholipid complex.
Factor X is a glycoprotein with a molecular weight of about 59,000 to about 70,000 and is composed of two subunits, one subunit having a molecular weight of about 40,000 and the other, about 19,000. Factor X is activated to Factor X.sub.a in both the intrinsic and extrinsic pathways.
Factor V, which has a molecular weight of 330,000, is a glycoprotein composed of one polypeptide chain containing a single, tightly-bound Ca.sup.2+ that is essential for the activity of V.sub.a. Factor V.sub.a is not a proteinase but acts as an accessory protein for prothrombin activation, which acts to increase the rate of prothrombin activation. Factor V itself is activated by thrombin; therefore, in a thrombin activation reaction, generated thrombin activates Factor V to V.sub.a, which in turn increases the rate of thrombin synthesis.
Studies in vitro show that Ca.sup.2+ and phospholipids increase the rate of conversion of prothrombin to thrombin by Factor X.sub.a by about 50 times as compared to the rate of conversion by Factor X.sub.a alone. Factor V.sub.a increases the rate of conversion by X.sub.a about 350-fold, but all factors together increase the rate at least about 20,000-fold. Thus, the Ca.sup.2+ -phospholipid-X.sub.a -V.sub.a complex produces the same amount of thrombin in one minute as would be produced by Factor X.sub.a alone in two weeks.
Since thrombin is the active agent in the conversion of fibrinogen to fibrin, the insoluble protein which forms blood clots, thrombin has applications in preventing blood loss from wounds, bleeding ulcers, surgical procedures, and other such injuries. Natural blood clotting requires several minutes to effectively stop the flow of blood from a site of injury; however, the use of thrombin at the site of injury results in immediate clotting. Therefore, topical application of thrombin is desirable in conditions existing after surgery and trauma, or oral application in the treatment of ulcers, to prevent life-threatening, massive blood loss. Also, thrombin infusion into the vitreous cavity during eye surgery can prevent bleeding from sites which are difficult to identify for cauterization. Such treatment reduces the risk of injury to the eye due to an increase in the intraocular pressure during routine surgery.
Currently, bovine thrombin preparations are available for topical applications. However, bovine thrombin preparations often lead to undesirable immunological reactions in patients being treated. Such reactions can be avoided by the use of human thrombin preparations. However, preparations of human thrombin are expensive, thus reducing the desirability of their use. Therefore, there is a need for a cost-effective means of purifying and activating prothrombin to thrombin from human sources.